Renal sodium homeostasis, a major determinant of blood pressure, is regulated by a variety of endocrine, autocrine, and neural factors. The molecular mechanism by which these factors act is not well understood but involves regulation of the rate of tubular sodium reabsorption. Several lines of evidence indicate that regulation of Na+,K+-ATPase activity is, at least in part, responsible for the reduced sodium reabsorption induced by dopamine in renal proximal tubules. At the cellular level, dopamine triggers a specific signaling cascade that ultimately leads to endocytosis of membrane Na+,K+-ATPase, a process that we have demonstrated is responsible for the decreased Na+,K+-ATPase activity. The aims of this proposal are: 1) To determine how the signal originated by phosphorylation of Na+,K+-ATPase alpha-subunit Ser-18 is transmitted to other domains of the a-subunit, such as the polyproline site (81-TPPPTTP-87) and the "endocytic sequence" (537-YLEL). 2) To test the hypothesis that PI3K binds to the Na+,K+-ATPase alpha-subunit polyproline site (81-TPPPTTP-87) and forms a scaffold for recruitment of other proteins that participate in the endocytosis of Na+,K+-ATPase molecules. 3) To identify the location and size of the intracellular pool that exchanges Na+,K+-ATPase molecules with the plasma membrane during dopamine induced Na+,K+-ATPase endocytosis. 4) To identify the stage(s) within the dopamine signaling cascade that is/are sensitive to changes of intracellular sodium concentration. 5) To test the in vivo regulatory role of dopamine by producing transgenic mice that express Na+,K+-ATPase molecules that are resistant to regulation by dopamine. These studies will provide insight into the molecular mechanism by which dopamine promotes renal sodium excretion in proximal tubules. Alterations in the regulation of kidney sodium reabsorption are often found on patients with congestive heart failure, cirrhosis, renal failure, and hypertension. Better knowledge of the mechanism of regulation of proteins involved in renal sodium transport will facilitate the development of new approaches in the treatment of patients with these disorders.